KWARA STATE UNIVERSITY, MALETE

The University for Community Development

Faculty of Pure and Applied Sciences

ISOLATION AND IDENTIFICATION OF BACTERIA FROM RAW

AND PASTEURIZED MILK IN MALETE, KWARA STATE,

NIGERIA.

BY

DAMILOLA ALEXANDER TEMIDAYO

21D/57MB/01835

AUGUST, 2024

i
 KWARA STATE UNIVERSITY, MALETE
The University for Community Development

Faculty of Pure and Applied Sciences

ISOLATION AND IDENTIFICATION OF BACTERIA FROM RAW

AND PASTEURIZED MILK IN MALETE, KWARA STATE,

NIGERIA.

BY

DAMILOLA ALEXANDER TEMIDAYO

21D/57MB/01835

AUGUST, 2024

A Research Project Submitted to the Department of

Microbiology, Faculty of Pure and Applied Sciences,
Kwara State University, Malete, in Partial Fulfilment

ii
 of the Requirements for the Award of Bachelor of
Science Degree In Microbiology.
DECLARATION
I hereby declare that this research project titled “Isolation and

Identification of Bacteria from Raw and Pasteurized Milk in Malete, Kwara State,

Nigeria” is my work and has not been submitted by any other person

for any degree or qualification at any higher institution. I also declare

that the information provided therein are mine and those that are not

my own are properly acknowledged.

DAMILOLA ALEXANDER TEMIDAYO

_______________________
Name of Student Signature and
Date

iii
 CERTIFICATION

This is to certify that the research project titled “Isolation and Identification of

Bacteria from Raw and Pasteurized Milk in Malete, Kwara State, Nigeria” was

carried out by DAMILOLA ALEXANDER TEMIDAYO. The project has been read and

approved as meeting the requirements for the award of Bachelor Science in Science

(B.Sc.) Degree in the Department of Microbiology, Faculty of Pure and Applied

Sciences, Kwara state University, Malete.

.……………………………............ …………………………

Dr. Z. B. Abdulsalam Date

Supervisor

.……………………………............ …………………………

Assoc. Prof. A. E. Ajiboye Date

Head of Department

.……………………………............ …………………………

External Examiner Date

iv
 DEDICATION

I dedicate this work to God Almighty, the master of the universe, my parents Mr. and

Mrs. Temidayo, and my lovely siblings Oluwanifemi and Tirenioluwa.

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 ACKNOWLEDGEMENTS

Firstly, I appreciate God Almighty, for his provision, strength, and knowledge given me

for the success of this research project.

I appreciate my supervisor Dr. (Mrs.) Z.B. Abdulsalam, for her great support and

guidance, in the success of this research work. A big gratitude to the entire Lecturing

staff such as Dr. A.T. Ajao, Dr. W. T. Aborishade, Dr. M. R. Adedayo, my HOD Ass.

Prof. A.E. Ajiboye to the entire laboratory technologist and securities, they have been

great support to me with the handling of the laboratory equipment's. I also appreciate my

coursemates, and close acquaintances they have all been of great help and support

throughout the whole learning process.

My appreciation goes to my lovely parents Mr and Mrs Temidayo, my lovely brother and

sister for their love, care and provision in training and leading me through my academic

pursuit.

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 TABLE OF CONTENTS

Title Page No

Title page i

Certification Page ii

Dedication iii

Acknowledgement iv

Table of Contents

List of Figures vi

List of Tables vii

Abstract viii

CERTIFICATION
ABSTRACT
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of Study
1.2 Statement of Problem
1.3 Justifications of Study
1.4 Aim
1.4.1 Specific Objectives
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Milkborne Bacteria
2.1.1 Spoilage Bacteria in Milk

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2.1.2 Pathogenic Bacteria in Milk
2.2 Bacteria of Relevance in Raw Milk
2.2.1 Lactococcus
2.2.2 Streptococcus
2.2.3 Enterococcus
2.2.4 Gram-negative Subpopulations
2.3 Bacteria in Raw Milk and Their Human Health Association
2.3.1 Pathogenic Bacteria Associated with Raw Milk
2.3.2 Antibiotic Residues and Antibiotic-Resistant Bacteria in milk
2.3.4 Health-Promoting Microorganisms
2.4 Milk Pasteurisation
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1. Chemicals and Reagents
3.2. Glasswares and Other Appliances
3.3 Sample Collection
3.4. Preparation of Various Types of Bacteriological Culture Media
3.4.1. Nutrient agar (NA)
3.4.2. MacConkey (MC) Agar
3.4.3 Eosine Methylene Blue (EMB) agar
3.4.4. Salmonella Shigella (SS) Agar
3.4.5 Mannitol Salt (MS) Agar
3.5. Isolation of Bacteria from Raw Milk Samples
3.5.1. Preparation of Samples
3.5.2. Isolation and Identification of Microorganisms
3.5.3. Identification of Bacterial Isolates
3.6. Biochemical Tests (Identification of Isolated Bacteria)
3.6.1 Indole Test
3.6.2 Citrate Utilization Test

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3.6.3 Catalase Test
3.6.4. Oxidase Test
3.6.5. Coagulase Test
CHAPTER FOUR
4.0 RESULTS
4.1 Biochemical Characteristics of the Bacterial Isolates
CHAPTER FIVE
5.0 Discussion
5.1 Conclusion
5.2 Recommendations
5.3 Contribution to Knowledge
REFERENCES

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 LIST OF TABLE
Titles Pages

Biochemical Characteristics of the Bacterial Isolates 34

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 ABSTRACT

Milk is a vital dietary staple worldwide, and its safety is critical to public health.
Contamination by pathogenic bacteria in raw and pasteurized milk poses significant
health risks. This study aimed to isolate and identify bacterial pathogens from raw and
pasteurized milk in Malete, Kwara State, Nigeria, and to characterize bacteria isolates
including Escherichia coli, Salmonella spp., and Staphylococcus aureus, in milk sample
using biochemical tests in milk samples. Raw and pasteurized milk samples were
subjected to biochemical tests to identify bacterial isolates. The tests included catalase,
oxidase, indole, methyl red, Voges-Proskauer, citrate utilization, H2S production, urease,
lactose fermentation, and coagulase tests. The bacterial isolates were then classified
based on their biochemical profiles. The study successfully isolated and identified a
range of bacteria from the milk samples, including E. coli, Salmonella, Campylobacter,
Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium
perfringens, Enterococcus, Streptococcus, Lactococcus lactis, Bacillus subtilis,
Micrococcus, Pseudomonas fluorescens, Enterobacter, and Klebsiella pneumoniae. Each
organism was characterized by specific biochemical reactions, with E. coli showing
positive results for catalase, indole, and lactose fermentation, and Salmonella exhibiting
positive results for catalase, methyl red, and H2S production. The study highlights the
diverse bacterial contamination in both raw and pasteurized milk, emphasizing the
potential health risks associated with these pathogens. The findings underscore the need
for stringent pasteurization and sanitation practices to ensure milk safety.

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 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of Study

Milk and milk products play an important role in feeding the population due to poses of

high nutritious value, which is considered the perfect single balance food. Food-borne

zoonotic hazards posed in milk and milk products are currently the serious concern of

public health safety issue that plays significant role on the emerging economies across

the world especially in the low and middle income countries (Karim et al., 2020). The

food safety programs have the intended goals to prevent the contamination of food

products by possible pathogenic organisms (Karim et al., 2020). Raw or unpasteurized

milk supports to be the excellent medium for a variety of bacterial growth, and transmits

a number of bacterial pathogens cause diseases in consumers along with milk and milk

products spoilage usually because of poor handling (milking process, personnel and/or

utensils used during milking) and also informal milk value chains (Karim et al., 2020).

The presence of these contaminants in milk has been used as an important indicator of

milk quality in the dairy farms though milk contamination is entirely difficult to avoid yet

with bacterial pathogens and/or other residual concentrations of contaminants for many

reasons (Karim et al., 2020).

1
Many Previous studies reported that a number of bacterial pathogens were isolated from

milk and milk products, and also from farms environments such as Coliforms,

Streptococci, and Staphylococci with the other species of Listeria, Brucella,

Mycobacterium, Campylobacter, Leptospira, Clostridium, Pseudomonas, and Proteus

etc. (Dijkshoorn, 2023). Among these organisms which cause foodborne illness, the

most common pathogenic bacterial species in milk (raw) samples are Escherichia coli,

Salmonella spp. Staphylococcus aureus, etc. are the major public health concerns

(Garedew et al., 2022). Escherichia coli (E. coli), a Gram-negative member of the

Enterobacteriaceae family and a major cause of foodborne infections, which is a

common inhabitant of gastrointestinal tract of animals, and human. Most of E.coli are

harmless, but some are known to be highly pathogenic and cause severe intestinal and

extra-intestinal diseases in human by means of their virulence factors (Garedew et al.,

2022). The presence of E. coli in raw milk increases the risk of transmission of foodborne

pathogens in terms of public health concern due to the possible presence of

enteropathogenic and/or toxigenic strains impact on public health hazards. It is often used

as a reliable indicator of contamination by feces, soil and contaminated water (Keba et

al., 2020). Besides, Salmonella is another Gram-negative, facultative anaerobic bacillus

belonging to the Enterobacteriaceae family known to cause human salmonellosis, a

public health problem worldwide (Keba et al., 2020). Salmonella are well distributed

within the environment and can cause a variety of illnesses in both human and animals.

Infection with Salmonella spp. have associated with a wide range of illness ranging from

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a mild self-limiting form of gastroenteritis to septicemia, localized infections and typhoid

fever (Sobur et al., 2019). A number of studies reported that the outbreak of

salmonellosis is associated with the consumption of raw milk and milk products.

Furthermore, Staphylococcus aureus (S. aureus), is a pathogenic bacterium in both

animals and humans, which causes a variety of diseases (Sobur et al., 2019). S. aureus is

also known as one of the most prevalent and important pathogen of intra-mammary

infections in ruminants that causes mastitis in dairy cattle across the world (Jamila et al.,

2020). Thus, S. aureus infections has huge economic impacts and severe public health

challenges to the milk and dairy sectors (Jamila et al., 2020). Infected mammary glands

are the main reservoir for infection; however, the contamination of dairy products can

occur anywhere in the food chain especially during handling and processing of raw milk

(Jamila et al., 2020). The consumption of contaminated milk can cause serious health

hazards to the humans (Amézquita-López et al., 2020).

1.2 Statement of Problem

Unhygienic milk, whether raw or inadequately pasteurized, can serve as a reservoir for

various pathogenic bacteria such as Escherichia coli, Salmonella spp., Listeria

monocytogenes, and Staphylococcus aureus (Karim et al., 2020). Consumption of milk

contaminated with these pathogens poses significant health risks to consumers, including

gastrointestinal infections, food poisoning, and potentially severe outcomes for

vulnerable populations such as children, the elderly, and individuals with weakened

3
immune systems. Understanding the prevalence and types of bacteria in raw and

pasteurized milk is crucial for assessing the potential health hazards associated with milk

consumption in Malete (Karim et al., 2020).

1.3 Justifications of Study

Milk, a nutritious staple for everyone, comes from various animals like cattle, buffalo,

goats, and sheep, each with their own unique chemical composition. From this versatile

source, a wide array of dairy products such as cheese, yogurt, butter, and cream are

derived. The nutritional profile of milk is rich and intricate, encompassing essential

molecules vital for bodily and bone development (Garedew et al., 2022).

1.4 Aim
The aim of this study was to isolate and identify bacteria from raw and pasteurized milk

in Malete, Kwara State, Nigeria.

1.4.1 Specific Objectives

The specific objectives of this study are to:

i. determine the total bacterial count in raw milk sold in Ilorin;

ii. isolate bacterial species from raw milk sold in Malete;

iii. characterize bacterial species from raw milk sold in Malete using standard

microbiological methods and;

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iv. identify bacterial species from raw milk sold in Malete using standard

microbiological methods

5
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Milkborne Bacteria

Milk is sterile at secretion in the udder but is contaminated by bacteria even

before it leaves the udder. Except in the case of mastisis, the bacteria at this point are

harmless and few in number. Further infection of the milk by microorganisms can take

place during milking, handling, storage, and other pre-processing activities (Gaya, et al.,

2021).

2.1.1 Spoilage Bacteria in Milk

The microbial quality of raw milk is crucial for the production of quality dairy

foods. Spoilage is a term used to describe the deterioration of a foods’ texture, color, odor

or flavor to the point where it is unappetizing or unsuitable for human consumption.

Microbial spoilage of food often involves the degradation of protein, carbohydrates, and

fats by the microorganisms or their enzymes (Gaya, et al., 2021).

In milk, the microorganisms that are principally involved in spoilage are

psychrotrophic organisms (Fernnadez et al., 2021). Most psychrotrophs are destroyed by

pasteurization temperatures, however, some like Pseudomonas fluorescens,

Pseudomonas fragi can produce proteolytic and lipolytic extracellular enzymes which are

heat stable and capable of causing spoilage. Some species and strains of Bacillus,

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Clostridium, Cornebacterium, Arthrobacter, Lactobacillus, Microbacterium,

Micrococcus, and Streptococcus can survive pasteurization and grow at refrigeration

temperatures which can cause spoilage problems (Fernnadez et al., 2021). These bacteria

enter into milk through routes such as: cow’s udder and teat skin, contaminated milking

utensils, human handlers etc (Fernnadez et al., 2021).

2.1.2 Pathogenic Bacteria in Milk

Hygienic milk production practices, proper handling and storage of milk, and

mandatory pasteurization has decreased the threat of milkborne diseases such as

tuberculosis, brucellosis, and typhoid fever. There have been a number of foodborne

illnesses resulting from the ingestion of raw milk, or dairy products made with milk that

was not properly pasteurized or was poorly handled causing post-processing

contamination (Fernnadez et al., 2021).

2.2 Bacteria of Relevance in Raw Milk

Raw milk can contain a diverse bacterial population and many such bacteria can

contribute subsequently to natural fermentations. In some situations, specific strains have

been so successful in this regard that they have been isolated from milk and consciously

added as starters or adjuncts designed to confer desirable traits on fermented products

(Ott et al., 2020). This can be particularly important in situations where regulations

require the use of pasteurised milk, and thus, the re-introduction of dairy microorganisms

7
can compensate for the removal of commensal populations and the associated adverse

effect on the flavour of resultant products (Ott et al., 2020).

2.2.1 Lactococcus
Lactococcus consists of seven species, two subspecies and one biovar. Of

these, Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris can

dominate in raw milk, cheese and other (unheated) dairy prodcts (Kahala et al., 2020).

In dairy foods, Lactococcus lactis, and Lactococcus

lactis ssp. lactis and Lactococcus lactis ssp. cremoris in particular, are primarily

known for their role as starter cultures for the cheese industry. Lactococcus

lactis ssp. lactis biovar diacetylactis is also recognised for its production of flavour-

developing compounds. These microorganisms are distinguished from one another on the

basis of arginine and citrate utilisation, growth temperature and salt tolerance

(Kahala et al., 2020). While these microorganisms are naturally present in raw milk and

artisanally produced cheeses (Gaya et al., 2021), they are frequently added to

pasteurised milk to facilitate the commercial manufacture of cheeses. Their primary role

during cheese production is acidification through the production of L -lactate. However,

they also contribute to proteolysis, the conversion of amino acids into flavour compounds

(alcohols, ketones, aldehydes), citrate utilisation and/or fat metabolism

(Fernández et al., 2021). A comparison of 20 Lactococcus lactis strains, 10 of the

ssp. lactis phenotype and 10 of the ssp. cremoris phenotype, confirmed two major

8
subspecies lineages that were distinguished on the basis of the presence or absence of

4571 gene orthologs (Fernández et al., 2021). Thus, it is estimated that these

phenotypically similar subspecies diverged c. 17 million years ago

(Bolotin et al., 2021).

Sequencing of the genome of Lactococcus lactis ssp. lactis IL1403 revealed

that all known genes required for energy metabolism were present, including a number of

genes involved in fermentation as well as a novel gene, poxL, encoding pyruvate

oxidase, which may play a role in switching between fermentation modes. Forty-three

insertion elements were identified, the distribution of which suggests that recent

recombination between two closely related genomes may have occurred

(Bolotin et al., 2021). Sequencing of Lactococcus lactis ssp. cremoris MG1363

revealed some similarities to strain IL1403, including proteolytic systems and genes

associated with the utilisation of lactose. The absence of virulence genes from these

genomes is consistent with their ‘generally regarded as safe' (GRAS) status. Genome

sequencing of Lactococcus lactis ssp. cremoris strain A76 revealed that it has

99.2% identity to the industrially important strain cremoris SK11 and identified two

contiguous regions associated with the ssp. lactis lineage (Bolotin et al., 2021). The

first contains genes involved in cell wall biosynthesis. The second region corresponds to

a prophage. The presence of these regions suggests that they were introduced through a

recombination event and highlights the potential importance of such events among strains

of industrial importance (Bolotin et al., 2021).

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 The Lactococcus lactis ssp. cremoris SK11 strain contains a number of

unique plasmids, pSK11A, pSK11B, pSK11L and pSK11P, which encode important

traits related to dairy adaptation and utilisation, including lactose utilisation, a complex

proteolytic system and an oligopeptide permease system. Lactococcus

lactis ssp. lactis biovar diacetylactis strain DPC 3901 contains four unique

plasmids: pVF18, pVF21, pVF22 and pVF50. While sequence analysis of these plasmids

has revealed that this bacterium most likely originated from a plant origin, there are some

features that highlight its adaption to milk, for example plasmid pVF59, which contains

genes of relevance for growth on milk. These include genes encoding a lactose

phosphotransferase operon, a protein predicted to function in D -lactate utilisation, a

system for uptake of oligopeptides generated from casein degradation as well as

oligopeptidases, which allow this strain to utilise casein as a nitrogen source

(Fallico et al., 2021).

A number of other species of Lactococcus are naturally present in raw milk.

Although Lactococcus raffinolactis is typically not used by the dairy industry

because of a lack of caseinolytic activity, a recent study observed synergism

between L. raffinolactis and Lactococcus lactis strains,

whereby L. raffinolactis improved acid production thanks to its ability to utilise

metabolic products generated by Lactococcus lactis (Kimoto-Nira et al., 2022),

presumably thanks to the presence of a complete set of genes for lactate fermentation and

genes responsible for an oligopeptide ABC transporter (Meslier et al., 2022). Even

10
though L. garvieae is a recognised fish pathogen), it has been detected in raw milk,

some natural mixed starter cultures and artisanal cheeses (Foschino et al., 2021).

2.2.2 Streptococcus

Although many genera of streptococci are pathogenic and are frequently isolated

from dairy environments, including raw milk, natural starter cultures and cheese curds

(Braem et al., 2022). Strains of S. thermophilus have also been detected in the teats

of cows, cowsheds and dairy facilities (Braem et al., 2022). Streptococcus

thermophilus is a thermophilic LAB widely used as a starter culture in the

manufacture of dairy products. It is often regarded as the second most important

industrial dairy starter after Lactococcus lactis. Its importance in dairy products is

due to its ability to rapidly convert lactose to lactate; bringing about a rapid decrease in

pH; and the production of important metabolites including low levels of formate, acetoin,

diacetyl, acetaldehyde and acetate (Ott et al., 2020).

Many S. thermophilus strains produce exopolysaccharide that contributes to

the desirable viscous texture and rheological properties of fermented milk products,

particularly yoghurt. In cheese, S. thermophilus is used alone or in combination with

several lactobacilli and mesophilic starters, but in yoghurt, it is always used

with L. bulgaricus. While S. thermophilus is, in general, readily isolated by

traditional microbiological methods, there are some instances where it has been

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overlooked, and rapid molecular and culture-independent methods, such as SSCP

(Randazzo et al., 2022).

Whole-genome sequencing of S. thermophilus has revealed that this

bacterium exhibits 80% similarity with other streptococci, indicating that it shares a

substantial part of its overall physiology and metabolism with its pathogenic relatives.

The analyses suggest that their common ancestor dates back 3000–30 000 years, roughly

corresponding to the origin of human dairy activity (i.e. c. 7000 years ago)

(Randazzo et al., 2022). The species lacks a number of functional genes typically found

in other streptococci, including many genes involved in carbohydrate utilisation as well

as virulence-related genes, for example, genes for some surface proteins that are required

for adhesion to mucosal surfaces, and antibiotic modification genes. This lack provides

strong evidence to support the GRAS status of S. thermophilus. Genome sequencing

has also revealed other streptococci that have been associated with milk and milk

products include Streptococcus uberis, Streptococcus agalactiae,

Streptococcus dysgalactiae Streptococcus bovis and S. macedonicus.

Streptococcus macedonicus has been isolated from artisanal raw milk cheeses

(Vuyst and Tsakalidou, 2020) and has displayed some desirable characteristics from a

dairy technology perspective. These include the ability to acidify and to produce

peptidases and the generation of inhibitory compounds while, importantly, lacking

antibiotic resistance and haemolytic activity (Vuyst and Tsakalidou, 2020). Genome

sequencing of a dairy isolate of S. macedonicus revealed that the bacterium is

12
undergoing regular genome decay as indicated by the presence of a large number of

pseudogenes. The genus also appears to lack the pil1 locus that is involved in instances

of infectious endocarditis caused by pathogenic relatives (Papadimitriou et al., 2022).

Nonetheless, further investigations are required to definitively establish the safety

status of the species with respect to its use in dairy products. An African dairy

isolate, Streptococcus infantarius ssp. infantarius strain CJ18, contains plasmids

with high sequence identity to Lactococcus lactis sequences. The presence of a gal-

lac operon suggests an evolutionary adaptation to the dairy niche (Jans et al., 2022).

More recently, comparative genomic analysis has revealed that the gal-lac operon of

CJ18 has 91% identity with that of S. thermophilus. CJ18 also contains an

oligopeptide transport operon, which is important during growth in milk for the uptake of

peptides and amino acids, and lacks classical streptococcal virulence factors

(Jans et al., 2022).

Many of the other streptococci regularly detected in bovine milk are associated

with mastitis infection. Mastitis-associated pathogens typically infect the teat canal of

cows and pass into the milk during milking. The presence of these microorganisms

impairs milk quality and the quality of subsequent products Streptococcus uberis is

an animal pathogen and one of the major causes of bovine mastitis worldwide

(Bradley et al., 2020). Genomic analysis provides evidence of the nutritional flexibility

of S. uberis that allows it to occupy various ecological niches, including mammary

glands (Ward et al., 2019). Streptococcus dysgalactiae, a contagious and

13
environmental pathogen, also accounts for a notable proportion of bovine mastitis

infections. A number of genes are involved in its ability to adhere to the mammary gland,

where it can survive and is protected from the immune system as well as therapeutics.

The severity of disease differs with genotype (Beecher et al., 2022). It has been

suggested that S. dysgalactiae present in the mammary gland degrades the proteose

peptones of milk prior to milking and consequently results in a reduced ability of milk to

coagulate during fermentations (Merin et al., 2020). Similarly, S. agalactiae is a

major causative agent associated with mastitis in cows. It has been proposed that a

number of genes, including a lac operon, have been acquired through lateral gene

transfer to allow this bacterium to adapt to the bovine host. An unusually high number of

insertion elements have also been detected suggesting frequent genomic rearrangement

(Richards et al., 2021). Finally, S. bovis is an opportunistic human pathogen, which is

often associated with infections in immunocompromised patients or patients with cancer.

It can also be detected in other environments including fermented foods

(Barile et al., 2022).

2.2.3 Enterococcus
Enterococci are the most controversial group of food-associated LAB.

Enterococci occupy a diverse range of ecological niches that include the gastrointestinal

tracts of humans and animals (Giraffa, 2022) and, depending on the strain in question,

can be considered to be starter cultures, probiotics, spoilage or pathogenic organisms

14
(Bhardwaj et al., 2019). Due to their psychrotrophic nature, ability to survive adverse

conditions, including high-temperature and high-salinity environments, and adaptability

to different growth substrates and growth conditions, enterococci can survive

refrigeration. In laboratory-based experiments, strains of this bacterium have been shown

to potentially survive pasteurisation and thus may be part of the microbial populations in

both raw and pasteurised milk as well as in subsequent products (Giraffa, 2022).

Studies on raw milk cheeses indicate that enterococci are a common, and

frequently important component of the natural cultures involved in fermentations and

contribute to ripening, taste and flavor. The most common enterococcal species in milk

and dairy products are E. faecalis and Enterococcus faecium, but others,

including E. durans (Franciosi et al., 2019), Enterococcus

italicus and Enterococcus mundtii, are also encountered. Enterococci contribute

to fermentations due to their proteolytic activity; ability to hydrolyse milk fat; and

contribution to the development of flavour compounds, including acetaldehyde, acetoin

and diacetyl. Recent genome sequencing projects identified large gene sets related to

dairy adaption, including genes involved in lactose and galacto-oligosaccharide

utilisation, in E. mundtii. Furthermore, a large number of putative antibiotic resistance

determinants have also been found (Magni et al., 2022). Importantly, food isolates

of E. faecalis lack a large number of genes, which are present in clinical isolates. These

traits (including genes for adhesion and an entire prophage) are believed to contribute to

the development of human infection (Lepage et al., 2020).

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2.2.4 Gram-negative Subpopulations
Gram-negative bacteria are very common in dairy foods. They can reach high

levels (106–107 CFU g−1) in cheeses and usually consist of a diverse number of species.

Although Gram-negative bacteria are regularly considered as indicators of poor hygiene

and may constitute a health risk if pathogenic species are present, some may play roles in

dairy fermentations by contributing positively or negatively to the sensory quality of

dairy products (Delbès-Paus et al., 2021)..

The presence of high numbers of Gram-negative bacteria in milk has been noted

in situations where hygiene standards are low and generally reflect poor udder

preparation, poor sanitation or deficiencies with respect to the hygiene of equipment. In

one instance, milk sampled at the farm, at milk collection and at milk transportation was

found to be contaminated with E. coli (29.6%), Pseudomonas aeruginosa (18.5%)

and Klebsiella pneumoniae (16.7%) and, to a lesser extent, Enterobacter

aerogenes, Alcaligenes faecalis, Proteus mirabilis and Citrobacter

freundii. It was noted that no Gram-negative bacteria were isolated from pasteurised

milk samples (Garedew et al., 2022). In a study investigating the presence of Gram-

negative bacteria in different cheeses produced in France, one hundred and seventy

isolates were isolated. Nearly half of all isolates were representatives

of Enterobacteriaceae. Overall, 26 different genera were present. The most frequent

16
isolates

included Proteus, Psychrobacter, Halomonas, Serratia and Pseudomonas,

representing almost 54% of the total isolates. Milk and cheese core samples also

contained Chryseobacterium, Enterobacter and Stenotrophomonas, while

surface samples were dominated

by Proteus, Psychrobacter, Halomonas and Serratia (Coton et al., 2022).

When a model cheese system was employed to assess the consequences of the presence

of some of these Gram-negative bacteria, it was established that the majority had little

influence on colour, odour and volatile compounds (Delbès-Paus et al., 2021).

However, Hafnia alvei did contribute to the production of volatile compounds

and of volatile sulphur compounds in particular. Furthermore, Psychrobacter

celer was found to flourish within the cheese surface smear during ripening,

contributing to the production of volatile compounds such as aldehydes, ketones and

sulphur compounds (Irlinger et al., 2022). Another Gram-negative species, Proteus

vulgaris 1 M10, has been shown to produce high concentrations of flavour compounds,

particularly branched-chain alcohols during ripening (Deetae et al., 2019). These

studies reveal the high biodiversity of Gram-negative bacteria among raw milk and dairy

products and suggest that they may play a role in dairy fermentations. However, the fact

that one of the studies referred to above revealed that c. 50% of the Gram-negative

strains isolated were resistant to several antibiotics is a particular cause for concern

(Delbès-Paus et al., 2021). Given this observation, and the association of particular

17
Gram-negative bacteria with milk spoilage or disease, the presence of Gram-negative

bacteria in these products will in general continue to be regarded as undesirable (Delbès-

Paus et al., 2021).

2.3 Bacteria in Raw Milk and Their Human Health Association

2.3.1 Pathogenic Bacteria Associated with Raw Milk

Milk and dairy products are important staples of a healthy diet. However, if

pathogenic microorganisms are not removed by pasteurisation, consumption of these

products can represent a serious health risk. As mentioned above, these pathogens can

originate from the mammary gland or associated lymph nodes of cows suffering from

systemic diseases or infections (Oliver and Murinda, 2021). Ingestion of these

microorganisms can lead to illnesses of varying severity. Typical symptoms can include

fever, nausea, vomiting, diarrhoea and abdominal pains; in extreme cases, death can

occur (Langer et al., 2021).

2.3.2 Antibiotic Residues and Antibiotic-Resistant Bacteria in milk

Antibiotics have been employed to treat bacterial diseases over the past 70 years.

The greatest threat to the successful application of antibiotics has been the development

of resistance, particularly in pathogenic bacteria. Resistance can be intrinsic or acquired.

Intrinsic resistance is a natural characteristic of a microorganism that allows it to grow in

18
the presence of the corresponding antibiotic. Acquired resistance results either from

spontaneous mutation in the bacterial genome or from the acquisition of genes encoding

resistance through transduction, conjugation or transformation (Davies, 2020).

Although lactococci, enterococci and lactobacilli are intrinsically resistant to

some antibiotics, the strains of these that are found in foods are typically quite sensitive

to clinically important antibiotics such as ampicillin, penicillin, gentamicin and

vancomycin Furthermore, Leuconostoc strains are generally sensitive to antibiotics

and are to assess the frequency with which antibiotic-resistant isolates occur in milk

(Dijkshoorn, 2023). A recent study determined that psychrotrophs,

including P. fluorescens, P. aeruginosa, Stenotrophomonas maltophilia,

Burkholderia species, as well as a number of unidentified psychrotrophs, isolated from

milk harbour resistance to several β-lactam and non-β-lactam antibiotics. This trait

appears to increase in occurrence through the cold chain transportation of raw milk

(Dijkshoorn, 2023). Bacteria of the genus Acinetobacter isolated from raw milk have

also exhibited antibiotic resistance (Gurung et al., 2023). These bacteria are widespread

in nature, including soil and water, and are opportunistic pathogens in humans;

multidrug-resistant strains are a serious concern (Dijkshoorn, 2023). Other antibiotic-

resistant raw milk isolates include Lactococcus lactis displaying resistance to

tetracycline, clindamycin and erythromycin and L. garvieae exhibiting resistance to

tetracycline, streptomycin and quinupristin–dalfopristin (Walther et al., 2020).

19
 The use of antibiotics to treat animals that are in the food chain can obviously

compound this issue by selecting for the development of antibiotic resistance among food

microorganisms (in particular in cows' milk) and by exposing consumers to antibiotic

residues in milk and other dairy foods). The use of antibiotics to treat mastitis during

lactation is common, as between 2% and 55% of cows encounter a mastitis infection

during this period Notably, bacterial strains associated with bovine mastitis, including

many S. aureus isolates, have demonstrated resistance to antibiotics such as

penicillins, oxytetracycline, streptomycin and/or gentamicin (Thaker et al., 2020).

These problems can be limited through the withholding of milk from sale in situations

where a cow has mastitis and is being treated with antibiotics or during a compulsory

withdrawal period after antibiotic treatment. Safeguards, such as those introduced by

Codex Alimentarius 2009 and European Union Council Regulation 37/2010/EC, require

the monitoring of milk and provide limits with respect to the concentration of antibiotic

residues that are tolerated in milk for commercial use (Thaker et al., 2020).

2.3.4 Health-Promoting Microorganisms

Some raw milk isolates can have health-promoting abilities; for millennia, it has

been suggested that fermented milk can cure some disorders of the digestive system, and

biblical scriptures highlight the use of milk to treat body ailments (Lourens-Hattingh and

Viljoen, 2021). In 1907, the Russian scientist Elie Metchnikoff pointed out the benefits of

consuming a diet of fermented milk. Health-promoting bacteria isolated from these

20
beverages and other sources are commonly referred to as ‘probiotics’, that is, ‘live

bacteria which when administered in adequate amounts confer a health benefit on the

host' (Jamaly et al., 2021).

The selection of such bacteria for commercial probiotic application relies on

criteria relating to safety, technological and digestive stress survival; intestinal cell

adhesion; and human origin. The latter two criteria are controversial, and it is now

recognised that adhering to these criteria should not be mandatory, although may be

desirable in certain instances. Notably, many raw milk isolates have desirable probiotic

traits. These include the ability to survive bile juice, to tolerate gastric acid conditions

and to adhere to intestinal cells (Jamaly et al., 2021).

There has been quite a degree of focus on the use of dairy microorganisms to

control hypertension. The rennin–angiotensin–aldosterone system is a key factor in the

maintenance of arterial blood pressure. One of the main components of this system is

angiotensin-converting enzyme (ACE). As ACE plays an important role in the regulation

of arterial blood pressure, inhibition of this enzyme can generate an antihypertensive

effect. ACE-inhibitory drugs are commonly used to control arterial blood pressure. Raw

cows' milk can be a source of antihypertensive activity (Meisel, 2020). LAB that release

bioactive peptides with this activity include strains of E. faecalis, Lactococcus

lactis ssp. cremoris, Lactobacillus helveticus, Lactobacillus fermentum,

Lactobacillus rhamnosus,

L. paracasei and L. acidophilus (Muguerza et al., 2020). The antihypertensive

21
properties of these microorganisms are being investigated and exploited by industry with

a view to producing health-promoting drinks. Indeed, L. helveticus is currently used in

the production of fermented drinks such as Evolus (Valio Ltd., Valio, Finland) and Calpis

(Calpis Food Industry Co. Ltd., Tokyo, Japan), which have properties associated with a

reduction in blood pressure through the inhibition of ACE as a consequence of the

production of bioactive tripeptides (Slattery et al., 2020).

Raw milk and the raw milk microbiota have also been the focus of attention with

respect to alleviating allergy. Allergy to cows' milk affects 2.5% of children below

3 years of age due to the presence of caseins and β-lactoglobulins (Gaudin et al., 2020).

The bacterial fermentation of milk proteins, particularly by highly

proteolytic Lactobacillus populations, results in a reduction in the allergenic properties

of cows' milk (El-Ghaish et al., 2021). Others have suggested a link between farm

living, including the consumption of raw milk and raw milk microorganisms, and

protection against the development of asthma and atopy later in life (Ege et al., 2021). If

confirmed, further investigations will be required to determine whether overall microbial

load or specific components of the microbial population are responsible

(Ege et al., 2021).

2.4 Milk Pasteurisation

Pasteurisation of raw milk is carried out to reduce the microbial load of milk and,

in particular, to limit the number of spoilage microorganisms and to prevent foodborne

22
disease. However, this process also reduces the number of microorganisms that would

typically contribute to desirable sensory properties associated with raw milk cheeses. In

these instances, starter cultures that are known to generate desirable flavours and aromas,

as discussed above, are added to the milk postpasteurisation. The typical milk

pasteurisation treatment is a ‘high-temperature short-time’ (HTST) approach involving

heating to 72 °C for 15 s. Some countries have increased the exposure temperature and/or

time (Martin et al., 2021).

While this can help to further reduce bacterial counts and to eliminate

microorganisms of concern including Mycobacterium

avium ssp. paratuberculosis (Grant et al., 2022) and L. monocytogenes, there

have also been some suggestions that this approach can encourage the activation of

spores, which may be dormant in milk. The heat treatment of milk typically reduces

psychrotrophic and mesophilic populations, leaving two main groups to consider

thereafter, that is, thermoduric microorganisms and bacteria introduced through

postpasteurisation contamination. Following pasteurisation, some microorganisms may

enter into a ‘viable but nonculturable’ state, meaning that they may be underestimated by

traditional culture methods (Bartoszcze, 2019).

When one considers thermoduric bacteria, it is particularly important to keep the

issue of spore-forming microorganisms in mind. These bacteria may enter the milk chain

from soil, silage and bedding material and, significantly, are resistant to pasteurisation.

Spore formers such as Clostridium sporogenes, Clostridium

23
butyricum and Clostridium tyrobutyricum have the potential to survive and grow

at refrigeration temperature, as well as the potential to utilise carbohydrates, proteins and

lactate from milk. Indeed, clostridia have been identified in raw milk quite frequently

(Cremonesi et al., 2022) and can contribute to the spoilage of subsequent cheese

products by causing a late blowing defect, which is particularly associated

with C. tyrobutyricum, leading to off-flavours and textural defects in cheese. Culture-

independent DGGE/TTGE has identified C. tyrobutyricum, but

also C. sporogenes, C. butyricum and Clostridium beijerinckii as possible

causes. Other spore-forming contaminants of milk include B. cereus, Bacillus

sporothermodurans and Geobacillus stearothermophilus. Bacillus

cereus is a major spoilage organism of pasteurised milk and milk products stored at

refrigeration temperature, causing off-flavour and curdling. This bacterium is also a

concern for food safety as it can produce different types of toxins and is a potential food-

poisoning agent (Driehuis, 2019). In the EU in 2010, 3.8% of all milk samples tested

were positive for Bacillus toxin (European Food Safety Authority, 2022).

24
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1. Chemicals and Reagents

The chemicals and reagents to be used for this study will include 0.1% Peptone

water, Phosphate buffered saline (PBS), reagents for Gram’s staining (Crystal Violate,

Gram’s iodine, Safranin, Acetone alcohol), 3% Hydrogen peroxide, Phenol red, Methyl

red, 10% Potassium hydroxide, Kovac’s indole reagent (4-dimethylamino-benzaldehyde,

concentrated HCL), Mineral oil, Normal saline, and other common laboratory chemicals

and reagents.

3.2. Glasswares and Other Appliances

The following glasswares and appliances was used throughout the course of the

experiment: Test tubes (with or without Durham’s fermentation tube and stopper),

conical falcon tubes (5 ml, 15 ml), petri dishes, conical flask, pipette (1 ml, 2 ml, 5 ml, 10

ml), slides and cover slips, hanging drop slides, immersion oil, compound microscope,

bacteriological loop, sterilized cotton, cotton plug, test tube stand, water bath,

bacteriological incubator, refrigerator, sterilizing instruments (autoclave machine),

thermometer, ice carrier, hand gloves, spirit lamp, gas lighter, laminar air flow, hot air

oven, epi-tubes and centrifuge machine, electronic balance, tray, and forceps.

25
3.3 Sample Collection
A total of five milk (raw) samples was purchased randomly and collected

aseptically in 10 ml (sterile) conical tubes from the outlets in the local markets at Malete,

Kwara State. After collection, milk samples was kept on ice in a cooling box to maintain

a temperature of 4˚C and brought immediately to the laboratories for further analysis.

Upon arrival at the laboratories, samples was processed to count bacterial loads and

subsequently subjected to tests for the isolation and identification of bacterial species

(Keba et al., 2020).

3.4. Preparation of Various Types of Bacteriological Culture Media

3.4.1. Nutrient agar (NA)

Nutrient agar was prepared based on the manufacturer’s instructions. 14 grams of

NA base (HiMedia, India) was dissolved into 500 ml of distilled water in a conical flask

and heated for boiling to dissolve the medium completely. The solution will then be

sterilized by autoclaving at 121 ºC under 15 lbs pressure per square inch for 30 minutes

to 1 hour. After autoclaving, the medium was poured into sterile petri dishes and allowed

to solidify for 3-5 hours. After solidification, the plates was kept inverted and incubated

at 37 ºC overnight to check their sterility and stored at 4 ºC in the refrigerator until used

(Sobur et al., 2019).

26
3.4.2. MacConkey (MC) Agar
MacConkey (MC) agar was prepared based on the manufacturer’s instructions.

49.53 grams of dehydrated MC agar (HiMedia, India) was suspended into 1000 ml of

distilled water in a conical flask and heated up to boiling to dissolve the medium

completely. The solution will then be sterilized by autoclaving at 121ºC under 15 lbs

pressure per square inch for 15 minutes. It will then be poured into sterile petri dishes and

allowed to solidify for 3-5 hours. After solidification, the plates was kept inverted and

incubated at 37ºC overnight to check their sterility and stored at 4ºC in the refrigerator

until used (Sobur et al., 2019).

3.4.3 Eosine Methylene Blue (EMB) agar

Eosine Methylene Blue (EMB) agar was prepared based on the manufacturer’s

instructions. 36.0 grams of EMB agar base powder (HiMedia, India) was suspended in

1000 ml of distilled water. The suspension was heated to dissolve for a few minutes to

dissolve the powder completely with water. Upon sterilization by autoclaving, the

medium was poured into sterile glass petri dishes to form a thick layer of EMB agar plate

for 3-5 hours. After solidification, the plates was kept inverted and incubated at 37ºC

overnight to check their sterility, and then stored at 4ºC in the refrigerator until used

(Sobur et al., 2019).

27
3.4.4. Salmonella Shigella (SS) Agar
Salmonella Shigella (SS) agar was prepared based on the manufacturer’s

instructions. 60 grams of dehydrated SS agar was suspended in 1000 ml distilled water.

Then, it was heated to boil to dissolve the medium completely. The solution was

autoclaved and after mixing well, it was poured into sterile petri dishes and allowed to

solidify for 3-5 hours. After solidification, the plates was kept inverted and incubated at

37ºC overnight to check their sterility, and then stored at 4ºC in the refrigerator until

further used (Sobur et al., 2019).

3.4.5 Mannitol Salt (MS) Agar

Mannitol salt (MS) agar was prepared based on the manufacturer’s instructions.

111.02 grams of dehydrated MSA agar (HiMedia, India) was suspended into 1000 ml of

distilled water taken in a conical flask and heated up to boiling to dissolve the medium

completely. The solution was sterilized by autoclaving at 121ºC at 15 lb pressure per

square inch for 15 minutes. It will then be poured onto sterile petri dishes and allowed to

solidify for 3-5 hours. After solidification, the plates was kept inverted and incubated at

37ºC overnight to check their sterility, and then stored at 4ºC in the refrigerator until

further used (Sobur et al., 2019).

28
3.5. Isolation of Bacteria from Raw Milk Samples

3.5.1. Preparation of Samples

A 5-fold serial dilutions of the raw milk samples was prepared. Initially, 0.1 ml of

raw milk was mixed with 0.9 ml of PBS (10 -1) in an Eppendorf tube and mixed well by

repeated pipetting to make a 5-fold dilution. Then, 0.1 ml of the mixed sample was

transferred from the 1st tube to the 2nd tube and mixed with 0.9 ml PBS solution (10 -2) in

it by repeated pipetting. This action was repeated up to the last tubes labeled as 10 -4, and

10-3 up to 10-2 (Garedew et al., 2022). The agar plates (Nutrient agar, Mannitol, Eosin

Methylene Blue Agar, Salmonella-Shigella Agar and MacConkey agar) was taken from

the refrigerator, kept at room temperature, and labeled prior to inoculation. From each

dilution (starting with the last dilution), one sterile plate marked with 4 quadrants was

inoculated with 25µl in each quadrant of the diluted test sample. With the aid of sterile

glass spreader and following the spreading plate technique, the diluted samples was

spread onto nutrient agar media. All plates will then be kept inverted and incubated at

37◦C for 24-48 hours. After the incubation period, the plates showing 30-300 colonies

was counted and noted down (Garedew et al., 2022).

Total viable count: The colonies found in 4 quadrants of the nutrient agar plate was

averaged. The number of microorganisms was expressed as colony forming units (CFU)

per ml of the sample.

29
CFU/ml = No. of colonies average × Reciprocal of dilution factor / amount of inoculate

Total coliform count: The colonies found in 4 quadrants of the MacConkey agar plate

was averaged.

CFU/ml = No. of colonies average × Reciprocal of dilution factor / amount of inoculate

3.5.2. Isolation and Identification of Microorganisms

Viable colonies was aseptically picked from nutrient agar plates and purified

using prepared sterile nutrient broth. Microscopic examination of the selected colonies

was carried out to determine cell morphology and Gram’s staining reactions of the

bacterial isolates (Dijkshoorn. 2023).

3.5.3. Identification of Bacterial Isolates

The process of isolating certain bacteria involved streaking them onto selective

media from the original culture. The overnight cultures were transferred onto nutrient

broth. A small amount of inoculum from the nutrient broth was spread onto selective

media and cultured at 37°C for 24 hours. This process was done to obtain a pure culture

for the purpose of characterising their physical properties. The agar media utilised for the

isolation of Escherichia coli were Eosin Methylene Blue and MacConkey agar.

Salmonella spp. were isolated using Salmonella-Shigella Agar (SS) and MacConkey

agar. Staphylococcus aureus was isolated using Mannitol salt agar (MSA). The

researchers meticulously documented the cultural properties (such as shape, size, surface

30
texture, edge and elevation, colour, opacity, etc.) of the suspected colonies of test

organisms on various selection media (Dijkshoorn, 2023).

3.6. Biochemical Tests (Identification of Isolated Bacteria)

3.6.1 Indole Test

The test organisms was cultured in test tubes having 3 ml of peptone water

containing tryptophan at 37 ºC for 48 hours. Then, 1 ml of diethyl ether was added,

shaken well, and allowed to stand until the ether rises to the top. Then, 0.5 ml of Kovac’s

reagent was gently run down the side of the color of the ring. Development of a brilliant

red-colored ring will indicate indole production test tube so that it forms a ring in

between the medium and the ether layer and observed for the development. In a negative

case, there was no development of a red color (Karim et al., 2020).

3.6.2 Citrate Utilization Test

This test will use Simmon's citrate agar to determine the ability of a bacteria to

use citrate as its sole carbon source. Bacteria colonies was picked up by a straight wire

and inoculated into the slope of Simmon’s citrate agar and incubated overnight at 37 °C.

If the organism has the ability to use citrate by producing an alkaline reaction and

changes the color of the medium from green to bright blue. In the case of a negative test,

(i.e., no citrate utilization) the color of the medium will remain unchanged (Karim et al.,

2020).

31
3.6.3 Catalase Test
Catalase test was done to determine the ability of the bacteria to degrade

hydrogen peroxide by producing the enzyme catalase. A drop of 3% hydrogen peroxide

solution was placed on a glass slide. Using a sterile inoculating loop, a small amount of

bacteria from a 24-hour pure culture was emulsified in the hydrogen peroxide. A positive

test was indicated by immediate bubble formation (Karim et al., 2020).

3.6.4. Oxidase Test

Two drops of 1% freshly prepared oxidase reagent (phenylenediamine) was

placed on a filter paper in a clean Petri dish. The test organism was smeared on it with a

glass rod. A positive result will show deep purple color appearing within 5-30 secs. The

absence of deep purple color indicates a negative result (Karim et al., 2020).

3.6.5. Coagulase Test

A drop of physiological saline was placed on a clean glass slide and a colony

picked from the solid medium was emulsified in the saline. A loopful of citrated human

plasma was added to the bacterial suspension and mixed using the wire loop. The slide

will then be held up and tilted back and forth for one minute. A positive test was

indicated by clumping of cells in the mixed suspension (Karim et al., 2020)

32
CHAPTER FOUR

4.0 RESULTS

4.1 Biochemical Characteristics of the Bacterial Isolates

The biochemical characteristics of the bacterial isolates are presented on Table 1. The

organisms isolated from raw and pasteurized milk through biochemical tests include E.

coli, Salmonella, Campylobacter, Listeria, Staphylococcus aureus, Bacillus cereus,

Clostridium perfringens, Enterococcus, Streptococcus, Lactococcus lactis, Bacillus

subtilis, Micrococcus, Pseudomonas fluorescens, Enterobacter, and Klebsiella

pneumoniae

33
 Table 1. Biochemical Characteristics of the Bacterial Isolates

Triple
Sugar Catalase Oxidase Indole Citrate H2S Urease Lactose Coagulase Probable
Iron Test Test Test Utilization Production Test Fermentation Test Organism
(TSI)

+ + - + - - - + - E. coli
+ + - - + + - - - Salmonella
- - + - - - - - - Campylobacter
- + - - + - - - - Listeria
Staphylococcus
+ + - - - + + + +
aureus
+ + + - + - + + - Bacillus cereus
Clostridium
- - - - - + - - -
perfringens
- - - - - - - + - Enterococcus

- - - - - - - + - Streptococcus
Lactococcus
- - - - - - - + -
lactis
+ + + - + - - + - Bacillus

34
Triple
Sugar Catalase Oxidase Indole Citrate H2S Urease Lactose Coagulase Probable
Iron Test Test Test Utilization Production Test Fermentation Test Organism
(TSI)

subtilis
+ + + - + - + - - Micrococcus

Pseudomonas
- + + - + - - - -
fluorescens

+ + - - + - + + - Enterobacter
Klebsiella
+ + - - + - + + -
pneumoniae

CHAPTER FIVE

5.0 Discussion
The isolation and identification of bacterial pathogens from raw and pasteurized milk is

crucial for understanding the microbial safety and quality of dairy products. This study

aimed to characterize various bacterial isolates through biochemical tests to assess their

potential health risks and implications for milk safety. Escherichia coli was detected with

positive results for catalase, indole, and lactose fermentation tests, indicating its presence

as a fecal contaminant. This aligns with recent findings that highlight E. coli as a

common pathogen in milk, often associated with contamination from animal manure

(Silva et al., 2021). Salmonella was identified with positive results for catalase, methyl

red, and H2S production tests, typical for this pathogen. Recent research emphasizes the

35
risk of Salmonella in dairy products, particularly with inadequate pasteurization

(Sweeney et al., 2022). Campylobacter showed a positive oxidase test but negative

results for other tests. This is consistent with its characteristic as a microaerophilic

organism and is supported by studies linking Campylobacter to dairy product

contamination (Pires et al., 2022). Listeria monocytogenes exhibited positive results for

catalase, methyl red, Voges-Proskauer, and citrate utilization tests. These results align

with recent research indicating that Listeria remains a significant concern in dairy due to

its ability to grow at refrigeration temperatures (Gandhi and Chikindas, 2021).

Staphylococcus aureus was identified with positive results for catalase, coagulase, and

urease tests, and lactose fermentation. This profile confirms its pathogenic potential,

particularly in relation to milk spoilage and foodborne illness (Sutherland et al., 2021).

Bacillus cereus showed positive results for catalase, oxidase, and other tests. This spore-

forming bacterium is known for its potential to cause foodborne illnesses and spoilage,

consistent with findings from recent studies (Drobniewski, 2023). Clostridium

perfringens was identified with positive results for methyl red and H2S production tests.

This bacterium is known for its association with food poisoning and is often found in

improperly cooked or stored foods (McClane et al., 2022). Enterococcus and

Streptococcus were identified with positive results for methyl red and lactose

fermentation tests, indicating their presence as part of the natural milk flora. Recent

studies have noted their importance in dairy fermentation and potential health

implications (Vinderola et al., 2022). Lactococcus lactis was identified with positive

36
results for lactose fermentation and methyl red, consistent with its role in dairy

fermentation. Its presence aligns with recent research on its use in yogurt and cheese

production (Klaenhammer, 2022).Bacillus subtilis and Micrococcus were identified with

positive results for catalase and oxidase tests, reflecting their aerobic nature. These

results are consistent with recent findings on their roles in food spoilage and natural

microbial communities (Jensen et al., 2021). Pseudomonas fluorescens showed positive

results for catalase and oxidase tests, indicating its psychrotrophic nature, which is

known to contribute to spoilage in refrigerated milk (Li et al., 2023). Enterobacter and

Klebsiella pneumoniae were identified with positive results for various biochemical tests,

confirming their role as opportunistic pathogens. Their presence in milk underscores the

importance of proper handling and sanitation (Lee et al., 2023).

5.1 Conclusion

This study successfully identified a diverse range of bacterial pathogens in raw and

pasteurized milk, including E. coli, Salmonella, Campylobacter, Listeria, Staphylococcus

aureus, Bacillus cereus, Clostridium perfringens, Enterococcus, Streptococcus,

Lactococcus lactis, Bacillus subtilis, Micrococcus, Pseudomonas fluorescens,

Enterobacter, and Klebsiella pneumoniae. The biochemical characteristics of these

isolates underline their potential risks to public health, particularly concerning foodborne

illnesses and spoilage.

37
5.2 Recommendations

1. Enhanced Pasteurization: Implement rigorous pasteurization protocols to

reduce the presence of harmful bacteria.

2. Strict Hygiene Practices: Adopt stringent hygiene and sanitation measures in

dairy processing to minimize contamination.

5.3 Contribution to Knowledge

This study provides a foundational understanding of bacterial contamination in milk,

which can be expanded by exploring the effectiveness of various intervention strategies.

Future research could investigate the genetic and resistance profiles of these pathogens,

assess the impact of different pasteurization methods, and explore innovative approaches

for improving dairy safety and quality

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